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Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

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Abstract

Accurate quantification of transcripts using quantitative real-time polymerase chain reaction (qPCR) depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 (ACT-2), elongation factor 1 alpha (EF-1α), elongation factor 1 beta (EF-1β), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), β-tubulin (β-TUB), and 18S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene (Gβ) across tissue types was also examined and normalized to the expression of each or both of UBQ and β-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further functional genomics studies in this economically valuable marine bivalve.

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Abbreviations

qPCR:

quantitative real-time transcription PCR

C t :

threshold value

E :

PCR efficiency

Q :

relative quantities

RT-PCR:

reverse transcription polymerase chain reaction

ACT-2:

actin-2

18S rRNA:

18S ribosomal RNA

GAPDH:

glyceraldehyde-3-phosphate dehydrogenase

EF-1α :

elongation factor 1 alpha

EF-1β :

elongation factor 1 beta

UBQ:

ubiquitin

β-TUB:

β-tubulin

Gβ :

G protein β-subunit gene

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Authors and Affiliations

  1. State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, 361102, China

    Fei Pu  (濮菲) & Caihuan Ke  (柯才焕)

  2. College of Ocean and Earth Science, Xiamen University, Xiamen, 361102, China

    Fei Pu  (濮菲), Bingye Yang  (杨丙晔) & Caihuan Ke  (柯才焕)

  3. Xiamen Medical College, Xiamen, 361008, China

    Bingye Yang  (杨丙晔)

Authors
  1. Fei Pu  (濮菲)
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  2. Bingye Yang  (杨丙晔)
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  3. Caihuan Ke  (柯才焕)
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Correspondence to Caihuan Ke  (柯才焕).

Additional information

Supported by the National Natural Science Foundation of China (No. 41176113), the National Basic Research Program of China (973 Program) (No. 2010CB126403), the Changjiang Scholars Program for Innovative Research Team in Universities (No. IRT0941), and the Earmarked Fund for Modern Agro-Industry Technology Research System (No. nycytx-47)

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Pu, F., Yang, B. & Ke, C. Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata . Chin. J. Ocean. Limnol. 33, 838–845 (2015). https://doi.org/10.1007/s00343-015-4078-x

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